CN1292033A - Baculovirus expression system and method for high through put expression of genetic material - Google Patents

Abstract

The present invention provides a novel recombinant baculovirus expression system for expressing foreign genetic material in host cells. This expression system can be conveniently applied to an automated method for expressing exogenous genetic material in a high-throughput manner. In other aspects, the present invention describes novel automated methods for determining the function of exogenous genetic material by infection into a host using the recombinant baculovirus expression system of the present invention.

Description

Baculovirus expression system and method for high-throughput expression of genetic material

Field of the invention

Broadly, the present invention relates to the fields of molecular biology and genetics. In particular, the present invention relates to a novel baculovirus expression system for expressing foreign genetic material by transfection of insect cells.

Background of the invention

A large amount of sequence information describing the genetic code of many smaller organisms has been generated. Great success has been achieved in similarly describing the genetic makeup of higher organisms such as humans in a much faster manner than previously thought possible. There will no doubt be increased efforts to complete the sequencing of the genetic code of these organisms, especially those important to humans such as plants and other mammals. There is a particular need to develop rapid and accurate methods for determining the function of specific genes and introns for which sequence data have been described in order to utilize these sequence data. The first step in this effort is to express the gene product from the sequenced genetic material. A key technology involved in accomplishing this efficiently has the potential to utilize robotics that can process many samples of genes or gene products simultaneously. Furthermore, optimal methods for purifying expressed gene products can be used to further advance the technology.

Baculoviruses infect insect cells. Both prokaryotic and eukaryotic cells have been used to express exogenous genetic material. However, prokaryotic cells such as E. coli cells are suitable for expressing gene products only if the products of exogenous genetic material do not require post-translational modifications, such as glycosylation, phosphorylation, or signal peptide excision. Prokaryotic cells do not contain the machinery required for this post-translational modification. Therefore, it is particularly important to develop new and easy-to-operate recombinant expression systems in eukaryotic cells. Baculovirus-infected insect cells perform most eukaryotic post-transcriptional modifications (including phosphorylation, N- and O-linked glycosylation, acetylation, disulfide cross-linking, oligomer assembly, and subcellular localization) , and it has been used to prepare functional recombinant proteins from various foreign genes.

Genetically modified baculoviruses are widely used to produce recombinant proteins (Davies, Bio/technology 12:47-50 (1994)). Typical baculoviruses include, but are not limited to, Autographa californica, Trichoplusiani, Rachiplusia ou, Galleria mellonella, and Bombyx mori. Of these, Autographa californica is probably the most comprehensively characterized and most widely used. The polyhedrin protein of baculoviruses is expressed at very high levels in the natural viral life cycle and is not required in cell culture. This allows for its replacement with almost any heterologous nucleotide sequence of interest. Any heterologous nucleotides inserted to replace polyhedrin can themselves be controlled by the polyhedrin promoter and thus achieve equally high levels of expression.

The baculovirus genome contains approximately 130 kb of DNA, which is too large to be easily manipulated by conventional plasmid cloning techniques, making it challenging to manipulate at the molecular level. The traditional solution to this problem is to introduce a cassette containing both the foreign gene, the appropriate promoter and termination sequences by homologous recombination. This is accomplished by flanking the cassette on the plasmid vector with the viral DNA sequences that flank the viral genomic site to be inserted. The method is less than 1% efficient.

Cloning vectors have thus been generated using plaque purification in the past. See Smith et al., U.S. Patent 4,745,051 for some conventional procedures; Smith et al., U.S. Patent 4,879,236; Summers et al., U.S. Patent 5,169,784; Guarino et al., U.S. Patent 5,077,214; Kang et al. , US Patent 5,194,376; Matsuura et al., US Patent 5,229,293 and Murphy et al., US Patent 5,516,657, the disclosures of which are incorporated herein by reference. Recently however several different techniques have been developed to increase the efficiency of recombination into the baculovirus genome. The most successful approaches are described below.

The baculovirus genome has been reconstructed into a replicon that propagates in Saccharomyces cerevisiae. This is achieved by inserting a yeast autonomously replicating sequence (ARS) at the polyhedrin site of the baculovirus genome, together with the CEN (centriole) sequence (which acts as a mitotic centromere to ensure that the viral genome segregates at a stable low copy number) and URA3 A selection marker (to allow growth in uracil-free media) was achieved. In this way, the recombination of foreign genes initiated by the polyhedrin promoter can be accepted by yeast, and the resulting baculovirus genome can be extracted from yeast cells and directly transfected into insect cells as a cloning vector. Advantages of this approach include near 100% efficiency and the ability to manipulate genes whose products may be toxic to insect cells in a foreign host. However, a large number of manipulations are required, making this method cumbersome and complicated.

The baculovirus genome can also be reconstituted into a replicon that can propagate in Escherichia coli. Luckow et al. (J. Virology 67: 4566-4579) described for the first time that the baculovirus genome can be modified into a large plasmid for replication in E. coli in a method identical to that used in yeast, called " bacmid". This was achieved by recombining a small F replicon (which confers the ability of the baculovirus genome to replicate autonomously and to stably segregate at low copy numbers) into the polyhedrin site, and inserting a kanr selection marker. The target site for the Tn7 bacterial transposon can also be introduced as an in-frame insert in the lacZa sequence derived from the pUC-derived plasmid. Therefore, the bacmid can perform intra-allelic complementation with the defective β-galactosidase lacZDM15 of E. coli hosts such as DH 1 OB. However, E. coli DH 1 OB containing a bacmid with a foreign gene inserted at the Tn7 site will maintain lacZaˉ, allowing colonies containing recombinant bacmids to be screened visually. The overall strategy of the procedure is to carry out the recombination and selection steps in a heterologous host (here E. coli) before transferring the finished product into insect cells. This strategy has similar advantages to the yeast system, but also involves many steps.

In addition to reconstitution of the baculovirus replicon in a heterologous host considered suitable for selection of recombinants, an in vitro recombination reaction was developed to transfer the gene from the transfer vector to the polyhedrin site of the virus. Using the Cre recombinase of phage Pi and its substrate lovp, the gene transfer of this system can be realized by a single enzyme exchange reaction. The target baculovirus genome (vaclox) and transfer vector were modified to contain lox sites. This 34 nucleotide sequence directs the Cre enzyme to convert the two substrate DNA molecules into topologically disjoint recombination products. The efficiency of the reaction is about 70% stoichiometrically. The biggest advantage of this method is its simplicity, but the maximum efficiency is only about 70%.

In 1990, Kitts et al. (Nucl. Acids Res.) 18: 5667-5672 (1990) introduced a single restriction site (Bsu36I) at the polyhedrin site from a covalently closed circular double-stranded molecule Novel baculovirus DNA was derived from existing wild-type baculovirus DNA. Linearizing the baculovirus genome with this restriction site reduced the efficiency of viral DNA transfection into insect cells, but co-transfection with a transfer vector driving polyhedrin site recombination produced a 3-fold higher ratio of recombinant virus.

In this strategy, the baculovirus containing the Bsu36I site is called ACRP-SC (single restriction site), and this strategy also transfers the polyhedron promoter and terminator sequences of the foreign gene to be expressed and its own copy of two double exchange events. About 15%-25% of the progeny viruses produced by this co-transfection are recombinants, but it should be noted that this is caused by the reduction of the wild-type virus background, rather than the increase in the absolute number of recombinants.

Subsequent development of this system effectively combined it with (lacZ-based) viral selection and replication-based selection strategies. See Kitts et al. Biotechniques 14:810-817 (1993). Recombination occurred between the conventional transfer vector and a derivative of the wild-type genome (designated BacPAK6) containing lacZ at the polyhedrin site and two additional Bsu36I sites flanking the polyhedrin sequence. Digestion of the baculoviral DNA with Bsu36I enzyme not only linearized the molecule but also removed two genomic fragments, thereby destroying the open reading frame ORF-1629. This gene is required for baculoviruses, so with the removal of the two Bsu36I fragments from the genome, competent virus can only be reconstituted by recombination with the transfer vector, whereby the genome will restore the complete ORF-1629. Also, the recombinant virus will form white plaques on a background of blue plaques from BacPAK6 DNA.

This strategy produces recombinant virus with a frequency of 85%-99%. Much of the background was due to contamination with undigested BacPAK6 DNA, which is more infectious than the linearized form of this genome. Viruses useful for future work may be a small fraction of the total virus produced, which can be purified by conventional techniques (plaque assay). Therefore, this method combines simplicity and efficiency well, and will be our method of choice for transferring array cDNA to expression vectors.

A particular problem associated with baculovirus expression systems is that they cause apoptosis or lysis of infected cells. The baculovirus apoptosis resistance gene (p35 gene) has been shown to increase virus production in certain cell lines. The recombinant baculovirus carrying this gene can be amplified selectively (up to 10 ⁶ times) in a suitable host.

Although the handling associated with the baculovirus expression system provides a useful unification of high yield and reliability, purification of the product of interest from baculovirus-infected cells is no easier than with any other eukaryotic system. A number of expression vectors have been developed to synthesize target antigens fused to polypeptides for ease of purification. For example, protein A fusion protein can be purified by IgG affinity, polyarginine fusion protein can be purified by cation exchange method, polyhistidine fusion protein can be purified by using its property of chelating zinc ion, β-galactosidase fusion protein And other proteins fused with specific immunogenic components can be purified by immunoaffinity, and fusion proteins of β-galactosidase, maltose-binding protein and glutathione-S-transferase (GST) can be purified by substrate affinity . Other epitope tags such as those recognized by EE or Glu-Glu antipeptide antibodies may also be used. The antibody is directed against a polypeptide containing the major tyrosine phosphorylation site of the t-antigen in polyoma. Such epitope tags have been used extensively in a variety of recombinant applications. Several investigators have reported that over 90% of attempts to purify EE-attached proteins have been successful, with most reaching 50% purity without additional chromatographic steps (Jim Litts and Robin Clark unpublished data).

This epitope tag has been characterized and optimized in detail by NEmotope peptide scanning analysis (Mario Geysen, John Wang and Robin Clark unpublished data). Antibodies with moderate affinity (Kd-2X10-7) for the EE tag (tag) allow both efficient binding of proteins in crude lysates and rapid elution of tagged proteins as free peptides under non-denaturing conditions. Although the EE tag is usually placed at the N-terminus of the protein, it can also be recognized when placed at the C-terminus or internally. Antibodies to this tag can also be used in immunoprecipitation, immunofluorescence, and Western blotting. Furthermore, high levels of this antibody can be produced by EE hybridomas. It has been reported that a 10 L preparation routinely yields 3-5 g of purified antibody. Another advantage of the EE tag is that it contains a strong tyrosine phosphorylation substrate for protein kinases such as src. This tag can be directly labeled with a radioactive phosphate or detected with an anti-phosphotyrosine antibody.

SUMMARY OF THE INVENTION

In one aspect, the present invention describes novel baculovirus expression systems that are particularly suitable for expressing heterologous genetic material in a host. Typically, the system comprises one or more selectable marker genes, a promoter such as the polyhedrin promoter of the polyhedrin structural gene or a fragment thereof, a transcription termination sequence such as the transcription termination sequence of the polyhedrin structural gene or a fragment thereof, A cloning site, an optionally included tag, and an optionally included restriction site or sites are manipulated to accept exogenous genetic material. Examples of selectable marker genes include apoptosis suppressor genes such as AcNPV p35, which in many embodiments are located upstream of the polyhedrin promoter. Examples of tags include EE tag sequences. In a preferred embodiment, the baculovirus expression system also contains the ORF-1629 gene, which can be used as a second selection marker gene. The present invention describes a method for preparing recombinant baculoviruses in which a DNA segment containing foreign genetic material is preferably combined with two selectable marker genes into a single DNA segment, which is then co-transfected with the baculovirus DNA into an insect host cells, thereby producing recombinant baculoviruses that can be isolated by screening with selectable marker genes. Therefore, the present invention can be used to prepare recombinant baculoviruses without a cloning step, and thus is useful for automated high throughput expression systems.

In a second aspect, the present invention describes a method for preparing a recombinant baculovirus expression system, which includes the steps of: (a) providing a baculovirus transfer vector, which contains an apoptosis suppressor gene, a promoter such as a polyhedrin structure The polyhedrin promoter of a gene or a fragment thereof, a transcription termination sequence such as that of a polyhedrin structural gene or a fragment thereof, a cloning site operable to accept exogenous genetic material, an optionally included tag and an optionally included one or more restriction sites, and (b) a cloning site for ligation of foreign genetic material into the baculovirus transfer vector such that the foreign genetic material is under the control of a promoter.

In a third aspect, the present invention describes a method for expressing foreign genetic material, which includes the steps of: (a) providing a baculovirus transfer vector containing one or more selection marker genes, promoters such as polyhedrin The polyhedrin promoter of the structural gene or a fragment thereof, a transcription termination sequence such as the transcription termination sequence of the polyhedrin structural gene or a fragment thereof, a cloning site operable to accept exogenous genetic material, an optionally included tag and an optionally included one or more restriction sites, (b) linking the foreign genetic material into the cloning site of the baculovirus transfer vector so that the foreign genetic material is under the control of the promoter, and (c) will contain The host cell is transfected with a baculovirus transfer vector of exogenous genetic material. In preferred embodiments, the present invention is amenable to automation and thus is useful for high-throughput expression.

In a fourth aspect, the present invention describes a method for detecting the function of exogenous genetic material, which includes the steps of: (a) providing a baculovirus transfer vector, which contains one or more selection marker genes, promoters such as polyhedron The polyhedrin promoter of a protein structural gene or a fragment thereof, a transcription termination sequence such as the polyhedrin structural gene or a fragment thereof, a cloning site operable to accept exogenous genetic material, optionally included tags and optionally One or more restriction sites that comprise, (b) connect exogenous genetic material into the cloning site of this baculovirus transfer vector, make this exogenous genetic material under the control of promoter, (c) will contain The host cell is transfected with the baculovirus transfer vector of the exogenous genetic material, and (d) observing the resulting biological outcome. In preferred embodiments, the present invention is amenable to automation and thus is useful for high throughput analysis.

In a fifth aspect, the present invention describes genes and fragments thereof, nucleotide sequences and gene products obtained by the methods of the present invention. The present invention describes the expression of selected nucleotide sequences in a host organism. Those skilled in the art should be well aware that the gene products of these nucleotide sequences can be isolated and purified using techniques known in the art. These isolations and purifications can be optimized according to some of the methods described herein. These gene products may exhibit biological activities such as pharmaceutical activity and other similar functions.

Brief description of the drawings

Figure 1 is a general illustration of high-throughput protein functional analysis that can be performed according to the methods of the present invention. Selected cDNA clones can be amplified, optionally tagged, and transfected in array format into linearized recombinant baculovirus transfer vectors. The recombinant baculovirus expression vector can then be transfected into a suitable host cell such as an insect cell. Products of exogenous genetic material can be purified by any suitable purification method such as affinity methods. Then, optionally, the purified protein can be further analyzed to determine its biological activity and/or function.

Figure 2 shows the application of the present invention in expressing genetic material such as EST cDNA clones. Baculoviruses containing the p35 inhibitor of apoptosis gene, the polyhedrin promoter, the EE tag, and a cloning site under the control of the polyhedrin promoter can be linked to exogenous genetic material such as EST cDNA clones, allowing the genetic material to be highly efficient. horizontal expression. Then, host cells such as insect cells are infected with the baculovirus expression system containing the foreign genetic material to achieve product expression of the genetic material. EST cDNA clones can be amplified according to PCR methods well known in the art, and cloned to the cloning site of interest by performing appropriate manipulations. This method results in high transcription and high translation rates of exogenous genetic material, allowing high-level protein production and rapid analysis of gene function.

Figure 3 shows the enzymatic detection of recombinant proteins in 96-well plates using recombinant baculoviruses expressing β-glucuronidase (Gus). Sf9 cells were grown to mid-log phase on insect serum-free medium, and then seeded on 96-well flat-bottomed polystyrene tissue culture plates at a density of 40,000 cells/100 μl/well. The rGus virus was serially diluted in 10-fold increments. After adding virus dilutions to the plates and incubating for several days, X-glucuronide stock solution was added to each well. X-glucuronide is the histochemical substrate of β-glucuronidase, which produces a blue substance after hydrolysis. A blue color was visible in many wells within minutes of substrate addition, with an increase in rGus product in infections caused by low MOI, and a chromogenic reaction appeared in high dilution wells after 24 hours.

Figure 4 shows the results of the analysis of MilliporeMAIP N45 high-efficiency protein binding plates containing Imobilon-P membranes treated with Glu or GST-added proteins, treated with monoclonal anti-Glu Glu antibody and goat anti-mouse IgG-conjugated second Antibody for alkaline phosphatase binding assay. Alkaline phosphatase was tested using SigmaFast BCIP/NBT tablets. These data indicated that the concentration range for detection of purified Rho-E, E2F-EE and Mut1-EE was 0.1-2.0 μg/well.

Figure 5 shows a scanned image of a 96-well plate illustrating the efficient production of recombinant baculoviruses using the method of the present invention without any cloning steps. A marker gene (Gus) whose expression can be detected by a chromogenic reaction was cloned into an IMAGE library vector (Stratagenebluescript). Bacterial cultures of this clone were grown in 96-well plates and used directly for PCR amplification (from bacteria). The designed PCR primers can hybridize with the vector sequence and contain sequences for ligation of independent cloning (Ligation Independent Cloning, LIC). The resulting product was digested with T4 DNA polymerase, annealed and ligated into the recombination arms (also treated with T4 DNA polymerase for LIC). Sf9 cells seeded in 96-well plates were then co-transfected with the ligation product and linearized baculovirus (polyBSU2 cleaved with Bsu36I enzyme). The obtained virus was passaged 3 times in Sf9 cells to increase and normalize the virus titer. Cells from the last passage were stained with Magenta-gluc (substrate for colorimetric assay of GUS) to detect the presence of recombinant virus (shown in magenta). The image shows that 75 of 88 experiments contained recombinant virus.

Detailed description of the invention

In one aspect, the present invention describes novel baculovirus expression systems that are particularly suitable for expression of heterologous genetic material in insect hosts. Typically, the system comprises one or more selectable marker genes, a promoter such as the polyhedrin promoter of the polyhedrin structural gene or a fragment thereof, a transcription termination sequence such as the transcription termination sequence of the polyhedrin structural gene or a fragment thereof, A cloning site, an optionally included tag, and an optionally included restriction site or sites are manipulated to accept exogenous genetic material. Examples of selectable marker genes include AcNPV p35, which in many embodiments is located upstream of the polyhedrin promoter, and in other embodiments may be located downstream of a transcription termination sequence. Examples of tags include EE tag sequences, preferably the ORF-1629 gene.

In some embodiments the present invention describes recombinant baculoviruses produced by transfecting insect cells with ligated fragments comprising transfer vector elements, selectable markers and cDNA, as opposed to clonal transfer vector plasmids shown in the prior art . Thus, the baculovirus expression system of the present invention greatly simplifies existing methods by eliminating the step of plasmid cloning and allowing the expression of cDNAs that cannot be cloned in bacteria. In this embodiment, selectable markers located on different fragments can be joined to fragments containing the cDNA coding sequence. This allows screening for recombinant baculoviruses containing the cDNA of interest. Any method suitable for joining DNA fragments can be used for this purpose.

In the most preferred embodiment, the recombinant baculovirus expression system of the present invention comprises an apoptosis suppressor gene encoding p35 protein, a polyhedrin promoter, and a gene cloned into the baculovirus cloning site under the control of the polyhedrin promoter. Exogenous genetic material, tag sequences located upstream or downstream of the cloning site such as the EE tag, and the ORF-1629 gene thought to be important for baculovirus replication and/or infection. Furthermore, convenient restriction sites may be included upstream and downstream of all of these elements, although these sites need not be adjacent to any one particular element. These convenient restriction sites include, but are not limited to, XhoI and PvuII sites.

In a second aspect, the present invention describes a method for preparing a recombinant baculovirus expression system, which includes the steps of: (a) providing a baculovirus transfer vector containing one or more selection markers, promoters such as polyhedron The polyhedrin promoter of a protein structural gene or a fragment thereof, a transcription termination sequence such as the polyhedrin structural gene or a fragment thereof, a cloning site operable to accept exogenous genetic material, optionally included tags and optionally comprising one or more restriction sites, and (b) a cloning site for ligating the foreign genetic material into the baculovirus transfer vector so that the foreign genetic material is under the control of the promoter.

In some embodiments, the recombinant baculovirus expression system can be prepared in an automated or high-throughput manner. In particular, restriction enzymes can be used to remove operable parts of the baculovirus from the baculovirus genome, including apoptosis suppressor genes, promoters such as the polyhedrin promoter of the polyhedrin structural gene or fragments thereof, transcription termination sequences such as A transcription termination sequence of the polyhedrin structural gene or a fragment thereof, a cloning site operable to accept exogenous genetic material and optionally included tags.

In the embodiment of the present invention in which the recombinant baculovirus expression system is prepared in an automated manner, high yields can be obtained in a relatively short period of time. Using this technique, genetic material such as cDNA can be inserted into cloning sites in an array, thus providing recombinant baculoviruses containing many different inserts.

In a third aspect, the present invention describes a method for expressing exogenous genetic material, which includes the steps of: (a) providing a baculovirus transfer vector containing an apoptosis suppressor gene, a promoter such as a polyhedrin structural gene The polyhedrin promoter or a fragment thereof, a transcription termination sequence such as that of the polyhedrin structural gene or a fragment thereof, a cloning site operable to accept exogenous genetic material, an optionally included tag and an optionally included one or A plurality of restriction sites, (b) link the foreign genetic material into the cloning site of the baculovirus transfer vector so that the foreign genetic material is under the control of the promoter, and (c) will contain the foreign genetic material Substances of the baculovirus transfer vector to transfect host cells.

Generally, in preferred embodiments, the host cells are insect cells. Baculoviruses have been shown to be pathogenic to certain species of insect cells. Therefore, in a preferred embodiment, an apoptosis suppressing gene is provided in the recombinant baculovirus expression system of the present invention. According to some embodiments of the present invention, Sf9 has particularly proven to be a particularly reliable animal cell for the production of recombinant proteins.

Specifically, the cloning of foreign genetic material and the isolation and purification of its expression product can be carried out automatically. Technologies that allow parallel processing of samples are the combination of various organizational modes, especially the widespread use of 96- and 384-well microtiter plates and the increasing use of sample manipulation robots designed to perform rapid, systematic work in this organizational mode. For example, 96-well circular microtiter plates were originally developed specifically for the field of immunology, but Lawrence Livermore National Laboratory developed 384-well plates for genomic research. Also, early robots were designed for use with 384-well plates to facilitate genomic research. See Copeland et al., Nature 369:421-422 (1994).

With the development of normalization and automation, cloned samples of genomic DNA and complementary DNA (hereinafter referred to as cDNA) are increasingly being provided in this normalized array format. For example, Lawrence Livermore National Laboratory and Los Alamos National Laboratory, part of the Department of Energy's National GeneLibrary Project, provide cosmids containing inserts primarily from single human chromosomes isolated by flow sorting libraries and phage libraries. Recently, through I. LawrenceLivermore National Laboratory. M． A． G． E． Consortium (Integrated Molecular Analysis of Genomes and their Expression Consortium) has access to a large number of cDNA clones. See Lennon et al. Genomics 33: 151-152 (1996).

I． M． A． G． E． The consortium facilitates the use of shared arrayed cDNA libraries. Through a collaboration with the University of Washington (Hillier et al., Genome Research 6:829-845 (1996), the sequences of these publicly shared clones were deposited without restriction and delay in the public sequence database, which contains short sequence tags or Expressed Sequence Tag (dbEST). In dbEST, more than 500,000 human cDNA clones have been arranged and contain more than 500,000 sequences. Sequence clustering analysis shows that most of the 80,000 or so human genes are present Has been represented by clones from this database. See Hillier, supra; Aaronson et al., Genome Res. 6:829-845 (1996).

I． M． A． G． E． The majority of clones available in cDNA databases are derived from dT-guided mRNAs with an average length between 1-2 kb. Therefore, the database more comprehensively represents the 3' 1-2kb region of many mRNAs, and less provides the 5' region and coding sequence. Of the 4,200 human mRNAs compiled by the Human Non-Redundant mRNA Collection (NCBI), about 25% can be completely identified by I. M． A． G． E． Of the clones represented, 50% were partially represented, and less than 25% were cloned without any representation. Based on resequencing of these clones, the estimated overall error rate is currently about 9%. Thus, there is a 91% chance that a cDNA thought to be located at a particular location on a microtiter plate has indeed grown from that well.

It is especially desirable to use these cDNA clones to provide the exogenous genetic material of the present invention. Furthermore, it is fully conceivable that other biological sources and other libraries arranged in different ways may also be suitable for use in the methods of the invention without specific experimental requirements.

Despite the high yield and reliability of the manipulations associated with recombinant viral expression systems, purification of target products from baculovirus-infected cells is no easier than with any other eukaryotic system. A number of expression vectors have been developed to synthesize target antigens fused to polypeptides for purification. For example, protein A fusion protein can be purified by IgG affinity, polyarginine fusion protein can be purified by cation exchange method, polyhistidine fusion protein can be purified by using its property of chelating zinc ion, β-galactosidase fusion protein And other proteins fused with specific immunogenic components can be purified by immunoaffinity, and fusion proteins of β-galactosidase, maltose-binding protein and glutathione-S-transferase (GST) can be purified by substrate affinity . Other epitope tags such as those recognized by EE or Glu-Glu anti-peptide antibodies may also be used. The antibody is directed against a polypeptide containing the major tyrosine phosphorylation site of the t-antigen in polyoma. Such epitope tags have been used extensively in a variety of recombinant applications. Several investigators have reported that over 90% of attempts to purify EE-attached proteins have been successful, with most reaching 50% purity without additional chromatographic steps (Jim Litts and Robin Clark unpublished data).

This epitope tag has been characterized and optimized in detail by NEmotope peptide scanning analysis (Mario Geysen, John Wang and Robin Clark unpublished data). Antibodies with moderate affinity (Kd-2X107) for the EE tag allow both efficient binding of proteins in crude lysates and rapid elution of tagged proteins as free peptides under non-denaturing conditions. Although the EE tag is usually placed at the N-terminus of the protein, it can also be recognized when placed at the C-terminus or internally. This antibody can also be used for immunoprecipitation, immunofluorescence, and Western blotting. Furthermore, high levels of this antibody can be produced by EE hybridomas. It has been reported that a 10 L preparation routinely yields 3-5 g of purified antibody. Another advantage of the EE tag is that it contains a strong tyrosine phosphorylation substrate for protein kinases such as src. Such tags serve as useful analytical detection markers for high-throughput protein interaction analysis. This tag can be directly labeled with a radioactive phosphate or detected with an anti-phosphotyrosine antibody.

In a fourth aspect, the present invention describes a method for detecting the function of exogenous genetic material, which includes the steps of: (a) providing a baculovirus transfer vector, which contains one or more screening marker genes, promoters such as polyhedron The polyhedrin promoter of a protein structural gene or a fragment thereof, a transcription termination sequence such as the polyhedrin structural gene or a fragment thereof, a cloning site operable to accept exogenous genetic material, optionally included tags and optionally One or more restriction sites that comprise, (b) connect exogenous genetic material into the cloning site of this baculovirus transfer vector, make this exogenous genetic material under the control of promoter, (c) will contain The host cell is transfected with the baculovirus transfer vector of the exogenous genetic material, and (d) observing the resulting biological outcome. In preferred embodiments, the invention is amenable to automation and thus useful for high throughput analysis.

Those skilled in the art will readily understand that there are various methods available for determining the function of exogenous genetic material expressed in hosts such as insect cells. In one embodiment, determination of nucleic acid function can be performed by complementation analysis. That is, the function of the target nucleic acid can be determined by observing the replacement or improvement of the function of one or more endogenous genes caused by the introduction of the target nucleic acid. In a second embodiment, nucleic acid function can be detected by analyzing biochemical changes in the accumulation of substrates or products of enzymatic reactions according to any method known to those skilled in the art. In a third embodiment, nucleic acid function can be determined by observing phenotypic changes in the host by methods including morphological, macroscopic or microscopic analysis. In a fourth embodiment, nucleic acid function can be determined by observing changes in host biochemical pathways resulting from local and/or systemic expression of exogenous genetic material. In a fifth embodiment, nucleic acid function can be determined by observing the inhibition of gene expression in the cytoplasm of the host cell as a result of expression of exogenous genetic material using techniques known in the art.

One particularly useful method of testing gene function is to observe the phenotype of an organism expressing specific foreign genetic material. Useful biophenotypic traits that can be observed microscopically, macroscopically or otherwise include, but are not limited to, increased tolerance to pesticides, increased tolerance to extremes of cold, heat, drought, salt or osmotic stress, (for fungi, Bacteria or viruses) Increased disease resistance, production of enzymes or secondary metabolites, etc. Other examples include the production of important proteins or other products such as antibodies, hormones, drugs and antibiotics, etc. The phenotypic trait may also be a desired secondary metabolite.

One strategy that has been proposed to facilitate this work is to create a database of expressed sequence tags (ESTs) that can be used to identify expressed genes. The accumulation and analysis of expressed sequence tags (ESTs) has become an important component of genomic research. EST data can be used to identify gene products and thereby expedite gene cloning. In order to store and link the enormous amount of sequence information generated by ongoing sequencing efforts, several sequence databases have been constructed. It is particularly expected that the method of the present invention can be used to expedite the work of isolating functional genes and determining their biological function.

In a fifth aspect, the present invention describes genes and fragments thereof, nucleotide sequences and gene products obtained by the methods of the present invention. The present invention describes the expression of selected exogenous genetic material, including nucleic acid sequences, in a host cell. Those skilled in the art should be well aware that the gene products of these exogenous genetic materials can be isolated using techniques known in the art. These gene products may exhibit biological activities such as pharmaceutical activity and other similar functions.

The following definitions are provided to further clarify the invention. Specific definitions are not intended to be limiting, but rather to clarify the meaning of terms used herein.

Expressed Sequence Tags (ESTs): Relatively short unidirectional DNA sequences obtained from one or more ends of cDNA clones and their derived RNA. They can exist in the 5' or 3' orientation. ESTs have been shown to be useful for the identification of specific genes.

Expression: As used herein, the term is meant to include one or more of transcription, reverse transcription, and translation.

Gene: An independent nucleic acid sequence responsible for the production of one or more cellular products and/or for the performance of one or more intercellular or intracellular functions.

Host: A cell, tissue or organism capable of replicating exogenous genetic material such as nucleic acid and transfected by baculovirus or its transfer vector fragment. The term is intended to include prokaryotic and eukaryotic cells, organs, tissues or organisms as appropriate.

Transfer vector: A nucleic acid sequence operable to transfer exogenous genetic material into a host cell for subsequent transcription and/or translation of the exogenous genetic material. Baculoviruses and fragments thereof including linearized fragments especially fall within the scope of this term.

Examples of preferred embodiments

The following examples further illustrate the invention. These examples are only intended to illustrate the invention and should not be construed as limiting. These examples are particularly intended to demonstrate the automated expression of exogenous genetic material in insect host cells by means of recombinant baculovirus expression systems for rapid production of proteins and polypeptides and subsequent evaluation of their function.

Example 1 Selection and rearrangement of cDNA in 96-well format

We selected a set of genes for which cDNA clones were available. These clones were rearranged in a 96-well plate format. The rearrangement robot used by Lawrence Livermore National Laboratory can be used for this purpose. Clonal identity was confirmed by sequencing as needed. Some clones were seeded in multiple wells to initially estimate well-to-well reproducibility. Simultaneously initiate the data tracking process to obtain information on clone identifiers, sequence access numbers, passage numbers, etc. for each rearrangement plate. Selected clones also represent a mix of known and unknown clones. For known clones, both some complete and some partial representatives are included.

Embodiment 2 Amplification of array cDNA

We directly transfected the amplified cDNA ligated into a linear baculovirus expression system (transfer vector) containing the necessary recombination and expression elements. This minimizes the steps needed to automate the procedure and provides the maximum flexibility needed to manipulate a variety of cDNA clones.

Design and synthesize PCR primers suitable for each cDNA library vector. This primer should include restriction sites for direct ligation of transfer vector fragments. The cDNA insert is amplified using these primers, cut with appropriate restriction enzymes, ligated into the transfer vector fragment, purified if necessary, and transfected into insect cells. Supernatants from these transfected cells were passaged to generate high titer recombinant virus stocks. We also investigated the possibility of direct single-base overhang ligation using undigested PCR products. It is important to use the highest fidelity thermostable polymerase available and the minimum number of amplification cycles to reduce sequence non-fidelity.

To ensure that each cDNA insert is in the correct reading frame with respect to the start codon of the vector, an appropriate vector must be selected for each cDNA clone. This can be accomplished by programming the mechanical pipette to pick the correct vector based on analysis of the known 5' (EST) sequence of the cDNA. Embodiment 3 accepts and is suitable for the construction of the baculovirus transfer vector of amplified cDNA

A series of new expression vector series were constructed to clone cDNA fragments from existing cDNA libraries. These expression vectors take advantage of recent advances in insect cell expression. Recombinant baculoviruses can be produced by transfecting insect cells with ligated segments containing transfer vector components and cDNA inserts rather than cloned transfer vector plasmids. This greatly simplifies the method for obtaining recombinant baculoviruses by eliminating the step of plasmid cloning. These vectors should include unique restriction sites such as SfiI and NotI specific for the primers used to amplify the cDNA insert. Thus, the protein product expressed from these vectors will contain an EE tag at the N-terminus followed by 2-4 amino acids from the linker sequence.

The baculovirus apoptosis resistance gene (p35 gene) has been shown to increase virus production in certain cell lines. Clem et al., Science 254: 1388-1390 (1991). The recombinant baculovirus carrying this gene can be amplified selectively (up to 10 ⁶ times) in a suitable host. See Lerch et al., Nucleic Acids Res. 21: 1753-1760 (1993). We included this gene in our vector design but deleted it from the host virus strain to ensure that the percentage of recombinant baculovirus in our virus stocks remained close to 100%. More specifically, the p35 apoptosis suppressor gene was inserted upstream of the polyhedrin promoter. This provides an alternative method for screening recombinant viruses when this vector is used in combination with a baculovirus strain lacking the p35 gene. Otherwise these vectors are identical to the pAcoG and pAcOGS vectors from which they were derived.

These vectors were co-transfected with DNA from polyBsu2, a baculovirus strain developed by Cetus (Martha Stampfer and Robin Clark, unpublished). polyBsu2 contains three Bsu36I sites, and its cleavage will remove the essential ORF-1629 gene from the baculovirus genome. Therefore, this strain is very similar to the reported strains. Kitts et al., Biotechniques 14:810-817 (1993). The activity of Bsu36I-digested polyBsu2 DNA can only be restored by recombination with vector DNA containing the ORF-1629 gene. This line can be modified to inactivate the p35 apoptosis suppressor gene using standard recombinant methods.

Example 4 Preparation of recombinant baculovirus by direct transfection of linked DNA

Many aspects of insect cell culture can be optimized for the development of especially robotic approaches. For example, seeding of Sf9 host cells can be optimized based on the seeding density of cells in a 96-well plate.

In several experiments designed to mimic high-throughput and/or automated procedures, we employed Sf9 inoculum derived from mass cultures, grown on serum-free insect medium, and grown in 96-well at various densities. Cells are grown on tissue culture plates. We obtained seeding densities that produced reproducible exponential growth within 24 hours. Thus, this cultivation allows the generation of cultures under optimal conditions for recombinant baculovirus infection and co-transfection of the donor transfer vector with recipient baculovirus DNA.

Example 5

We will optimize methods for protein production from recombinant baculovirus expression systems. However, existing and literature-described methods are now considered acceptable. Miniaturization and automation of the necessary operations including cell culture, infection, harvesting of infected cells, preparation of lysates, affinity chromatography and storage of purified proteins can be optimized according to known procedures without special experimentation.

Example 6 Affinity Purification of Recombinant Protein Using Glu-Glu Tag

The product of interest can be purified from baculovirus-infected cells according to any of a number of methods. A number of expression vectors have been developed that allow the synthesis of target antigens fused to polypeptides for ease of purification. For example, protein A fusion protein can be purified by IgG affinity, polyarginine fusion protein can be purified by cation exchange method, polyhistidine fusion protein can be purified by using its property of chelating zinc ion, β-galactosidase fusion protein And other proteins fused with specific immunogenic components can be purified by immunoaffinity, and fusion proteins of β-galactosidase, maltose-binding protein and glutathione-S-transferase (GST) can be purified by substrate affinity .

Epitope tags recognized by EE or Glu-Glu anti-peptide antibodies can also be used. See Grussenmeyer et al., Proc. Natl. Acad. Sci. 82:7952-7954 (1985). The antibody is directed against a polypeptide containing the major tyrosine phosphorylation site of the t-antigen in polyoma. See Talmage et al., Cell 59:55-65 (1989). Cetus Corporation developed this tag for use in protein purification (Rubinfeld et al., Cell 65:1033-1042 (1991)), and it is also the primary tag used by Chiron Corporation for this purpose. This epitope tag has been well characterized and optimized by Mimotope peptide scanning analysis (Mario Geysen, John Wang and Robin Clark unpublished data). Antibodies with moderate affinity (Kd-2X10-7) for the EE tag allow both efficient binding of proteins in crude lysates and rapid elution of tagged proteins as free peptides under non-denaturing conditions. Although the EE tag is usually placed at the N-terminus of the protein, it can also be recognized when placed at the C-terminus or internally. This antibody can also be used for immunoprecipitation, immunofluorescence, and Western blotting. See Garcia et al., Mol. Cell Biol. 13:6615-6620 (1993). Since high levels of this antibody can be produced by EE hybridomas, the supply of this antibody is not a problem. A 10 L preparation routinely yields 3-5 g of purified antibody. Another advantage of the EE tag is that it contains a strong tyrosine phosphorylation substrate for protein kinases such as src. See Garcia et al., J. Biol. Chem. 268:25146-25151 (1993). This tag can be used as an analytical detection marker for high-throughput protein interaction analysis. This tag can be directly labeled with a radioactive phosphate or detected with an anti-phosphotyrosine antibody.

The use of this EE tag was facilitated by a series of baculoviral expression vectors called pAcOG1, 2 and 3, in which the original polylinker of pAcC13 had encoded the initial methionine, Glu-Glu Synthetic polylinker replacement for epitope tags and multiple unique restriction sites. See Munemitsu et al., Mol. Cell Biol. 10:597-5982 (1990). These vectors allow one-step construction of baculovirus expression vectors with an inserted coding sequence (with a natural stop codon) fused in-frame to an N-terminal EE tag. Another set of vectors, pAcO G1S, 2S, and 3S, also contain a series of tandem terminators in all three reading frames for incomplete constructs that do not themselves have a stop codon. The order of these sites is influenced by the most common restriction enzymes used to construct cDNA libraries. The vector itself is optimized for expression by including unique sequences upstream and downstream of the initial methionine that have been experimentally shown to enhance expression in Sf9 cells. These vectors have been used to prepare many EE-appended proteins.

In order to illustrate that proteins to which Glu has been added can be conveniently analyzed by immunoassay in a 96-well format suitable for high-throughput automated production, we performed the following experiments. Millipore MAIP N45 High-Performance Protein Binding Plates containing Immobilon-P filters were treated with Glu or GST-tagged proteins, and binding was assayed with monoclonal anti-Glu-Glu antibody and alkaline phosphatase conjugated goat anti-mouse IgG secondary antibody degree. Alkaline phosphatase was detected with SigmaFast BCIP/NBT tablets. As shown in Figure 4, the concentration range of detected purified Rho-E, E2F-EE and Mytl-EE was 0.1-2.0 μg/well. These results demonstrate that the Glu tag is a convenient and specific immunodetection target for quantification of recombinant proteins in a 96-well format. Moreover, the technique is easily applicable to limiting dilution experiments for screening recombinant baculoviruses and for in situ detection of recombinant proteins in 96-well tissue culture.

Example 7

These data demonstrate that enzymatic analysis of baculovirus-expressed recombinant proteins is feasible in the same 96-well format in which Sf9 cells were seeded and virus-infected. The virus we used expresses an enzyme (eg, Gus) that can be easily detected by a colorimetric assay. However, the same principles can be extended to proteins with other functions.

Enzymatic detection of recombinant proteins was performed in a 96-well format using a recombinant baculovirus expressing β-glucuronidase (Gus). Sf9 cells were grown to mid-log phase on serum-free insect medium, and then seeded on 96-well flat-bottomed polystyrene tissue culture plates at a density of 40,000 cells/100 μl/well. The rGus virus was serially diluted in 10-fold increments. Add virus dilutions to the culture plate and incubate for several days, then add X-glucuronide stock solution to each well. X-glucuronide is the histochemical substrate of β-glucuronidase, which produces a blue substance after hydrolysis.

A blue color was visible in many wells within minutes of substrate addition, with an increase in rGus product in infections caused by low MOI, and a chromogenic reaction appeared in high dilution wells after 24 hours. Chromogenic reactions were seen both in supernatants of infected cells and in single cells examined by microscopy. As shown in Figure 3, these results demonstrate that enzymatic activity can be readily detected in baculovirus-infected cultures infected at low MOI at low cell numbers. Thus, performing this and related in situ enzymatic assays following baculovirus infection may be amenable to high-throughput automation.

Example 8

This example illustrates some of the principles of the invention in which recombinant baculoviruses were produced from cDNA clones in a miniaturized (96 well) format without a cloning step. The basic elements of the invention are shown schematically in Figure 2 and discussed above.

Step 1: Arrange bacterial cultures containing cDNA cloning plasmids in 96-well plates. This plasmid is a Bluescript cDNA library vector containing the Gus gene commonly used as an expression marker.

Step 2: Using primers that hybridize to the plasmid vector sequences on both sides of the cDNA insertion site, the Gus gene insert is amplified by a PCR program. These primers also contained a restriction site at the 5' end (NotI for the 5' primer and SfiI for the 3' primer). The portion of the primer containing the restriction site does not hybridize to the plasmid vector sequence, but is incorporated into the PCR product.

Step 3: The PCR product from step 2 was digested with NotI and SfiI restriction endonucleases to create appropriate cohesive ends on the product.

Step 4: Purify the restriction fragments generated in step 3 by chromatography in an Arrayit 96-well DNA purification device.

Step 5: Digest pAcOP2 with NotI, SfiI, PvuII and XhoI restriction enzymes to generate baculovirus transfer vector arms. Plasmid pAcOP2 contains an EE epitope tag followed by a cloning site containing NotI and SfiI restriction sites. The EE tag includes a start codon positioned such that it can be transcribed by the polyhedrin promoter. pAcOP2 also contains the orf-1629 and p35 selection markers and is therefore an example of a baculoviral transfer vector as shown in Figure 2 and described above. DNA from the digested product was purified by chromatography on a Qiagen column.

Step 6: Mix the digested fragments from steps 4 and 5 and ligate with DNA ligase.

Step 7: Digestion of baculovirus DNA containing a Bsu36I restriction endonuclease site in the orf-1629 region with Bsu36I yields a linearized fragment lacking the entire orf-1629 gene.

Step 8: Co-transfect the ligated product of Step 6 and the baculovirus DNA of Step 7 into insect (Sf9) cells (seeded in a 96-well plate) using a standard insect cell infection procedure such as Invitrogen lipofection.

Step 9: Incubate the transfected Sf9 culture from step 8 for 5 days. The obtained baculovirus stock solution was passed twice on Sf9 cells.

Step 10: Dilute the passaged virus stock solution in step 9 10-fold with a medium containing 150ug/ml X-gluc, and use it to infect Sf9 cells in a 96-well plate. Eight of the 96 wells were used as experimental controls.

Step 10: Two days after infection, count the number of blue wells in the culture plate of step 10, which indicates the expression of the GUS gene. The percentage of blue wells relative to the total number of wells indicates the efficiency and success of this approach.

Claims (22)

1. A baculovirus expression system comprising at least one selectable marker gene, a promoter, a transcription termination sequence and a cloning site operable to accept foreign genetic material. 2. The baculovirus expression system of claim 1, wherein the promoter comprises the polyhedrin promoter of the polyhedrin structural gene or a fragment thereof. 3. The baculovirus expression system of claim 1, wherein the transcription termination sequence comprises the transcription termination sequence of the polyhedrin structural gene or a fragment thereof. 4. The baculovirus expression system of claim 1, further comprising a tag and one or more restriction sites. 5. The baculovirus expression system of claim 1, wherein the selectable marker gene is the apoptosis suppressing gene p35 gene. 6. The baculovirus expression system of claim 5, wherein the apoptosis suppressor gene is located upstream of the polyhedrin promoter. 7. A method for producing a recombinant baculovirus expression system, comprising the steps of: (a) providing a baculovirus transfer vector comprising at least one selection marker gene, a promoter such as the polyhedrin promoter of the polyhedrin structural gene or a fragment thereof, a transcription termination sequence such as the polyhedrin structural gene a transcription termination sequence or fragment thereof, a cloning site operable to accept exogenous genetic material, optionally containing a tag and optionally one or more restriction sites; and (b) Linking the foreign genetic material into the baculovirus transfer vector at the cloning site so that the foreign genetic material is under the control of the promoter. 8. 7. The method of claim 7, wherein said baculovirus transfer vector is linearized. 9. 7. The method of claim 7, wherein said exogenous genetic material is cDNA. 10. 7. The method of claim 7, wherein said linking is performed automatically in a multiwell array. 11. A method for expressing exogenous genetic material comprising the steps of: (a) providing a baculovirus transfer vector comprising an apoptosis suppressor gene, a promoter such as the polyhedrin promoter of the polyhedrin structural gene or a fragment thereof, a transcription termination sequence such as the polyhedrin structural gene A transcription termination sequence or a fragment thereof, a cloning site operable to accept exogenous genetic material, and optionally contain a tag and optionally one or more restriction sites; (b) linking the foreign genetic material into the baculovirus transfer vector at the cloning site so that the foreign genetic material is under the control of a promoter; and (c) Transfecting the baculovirus transfer vector containing the foreign genetic material into the host cell. 12. The method of claim 11, further comprising purifying the gene product produced. 13. The method of claim 12, wherein said purification is performed by immunoassay of the tag epitope. 14. The method of claim 11, wherein said baculovirus transfer vector is linearized. 15. The method of claim 11, wherein said exogenous genetic material is cDNA. 16. 11. The method of claim 11, wherein said linking is automated in a multiwell array. 17. The method of claim 11, wherein the host cell contains a baculovirus genome. 18. 11. The method of claim 11, wherein the host cell contains a baculovirus genome, with the proviso that the baculovirus genome does not contain an apoptosis-inhibiting gene. 19. A method for determining the function of exogenous genetic material, comprising the steps of: (a) providing a baculovirus transfer vector comprising an apoptosis suppressor gene, a promoter such as the polyhedrin promoter of the polyhedrin structural gene or a fragment thereof, a transcription termination sequence such as the polyhedrin structural gene A transcription termination sequence or a fragment thereof, a cloning site operable to accept exogenous genetic material, and optionally contain a tag and optionally one or more restriction sites; (b) connecting the foreign genetic material to the baculovirus transfer vector at the cloning site, so that the foreign genetic material is under the control of the promoter; (c) transfecting the baculovirus transfer vector containing the exogenous genetic material into the host cell; and (d) Observing the resulting biological consequences. 20. 20. The method of claim 19, wherein said observing comprises analyzing biochemical changes in the accumulation of substrates or products of enzymatic reactions. twenty one. 21. The method of claim 19, wherein said observing comprises comparing RNA expression in a first cell containing the exogenous genetic material to RNA expression in a second cell that does not contain the exogenous genetic material of interest. twenty two. A product selected from the group consisting of genes or fragments thereof, nucleotide sequences and gene products produced by the method according to claim 11.

Images